Project Summary Candida spp. are a frequent cause of bloodstream infections, with an estimated 50,000 cases and 15,000 deaths at a cost of $2 billion per year in the US. Previous research has largely focused on C. albicans, however non-albicans species such as C. parapsilosis, C. krusei, C. tropicalis, C. glabrata and C. auris are emerging as significant causes of systemic infection, especially in vulnerable populations. In many cases, these species have intrinsic resistance to current antifungal drugs which complicates their treatment. Given that relatively few antifungal agents are available and the current void in antifungal discovery, resistance is a major public health concern. Novel antifungal compounds are therefore urgently needed to address this challenge. Our long-term goal is to identify virulence mechanisms in C. parapsilosis which may be mitigated by such compounds. A key step in the virulence process of these organisms is adhesion to host tissues and this step requires specific adaptations. We have recently discovered that clinical isolates of C. parapsilosis grown under conditions that mimic a human host exhibit upregulation of several potential virulence factors, including Phr1 ? a fungal cell wall transglycosylase that is required for invasive isolates of C. parapsilosis to adhere to extracellular matrix proteins under fluid shear. We propose to elucidate the changes that occur at the cell surface during Phr1 induction and adhesion. The rationale that underlies this proposal is that these changes represent potential targets for novel antifungals that disrupt virulence mechanisms rather than inducing fungal death. Importantly, such antifungals are likely to be highly selective to fungi and broadly effective across pathogenic fungal species, while avoiding the selective pressure that would lead to emergence of resistance. Based on our preliminary data, our central hypothesis is that using synthetic inhibitors that target this transglycosylase and/or other cell wall glycoproteins that are associated with Phr1 expression will disrupt key steps in the pathogenesis pathway to limit infection. The project will accomplish the following specific aims: To further capture the complex changes in the cell wall glycoproteome in C. parapsilosis during Phr1 induction and to design glycosyl triazole inhibitors that disrupt C. parapsilosis adherence. The first aim will be accomplished through enzymatic release of cell wall glycoproteins and their identification through mass spectrometry while the second aim will be accomplished through informed design and synthesis of compounds which will be tested in established adhesion assays. The combination of Dr. Reid's expertise in glycoprotein biochemistry with Dr. Bliss' clinical and research expertise in Candida pathogenesis brings together the skills needed to test this idea and also provides a pipeline for student involvement and education on a ?real-life? application of biochemistry to a clinical problem. The knowledge gained from these studies will enhance understanding of the early stages of candidiasis that lead to colonization and ultimately disseminated infection in a non-albicans species, and will provide insights into strategies to alter this process.